Lymphohematopoietic differentiation from embryonic stem cells in vitro

grantor: University of TorontoThe 'in vitro' differentiation of embryonic stem (ES) cells provides a model system for the study of hematopoietic development. However, the extent to which ES cells can recapitulate 'in vivo' lymphopoiesis and facilitate the study of lymphohematopoietic differentiation remains to be determined. ES cells cultured on the bone marrow (BM)-derived stromal cell line, OP9 (ES/OP9 coculture), were reported to differentiate into B cells 'in vitro' but it was not known whether these phenotypic B cells were capable of functional responses. ES cell-derived B lymphocytes were shown to be functionally analogous to normal fetal liver (FL)- or BM-derived B lineage cells at different developmental stages, including response to the cytokine Flt-3 ligand at early stages, and response to mitogen at later stages. Although B cells could be efficiently generated from ES cells, it remained unknown whether ES cells could give rise to T lymphocytes ' in vitro'. ES cells, unfractionated ES cell-derived precursors, and ES cell-derived CD45+ hematopoietic precursors proved to be unsuitable progenitors for the generation of T lymphocytes in fetal thymic organ cultures (FTOCs). In contrast, ES cell-derived Flk1+ CD45- precursors reproducibly gave rise to T cells when transferred into FTOCs. The molecular events regulating the onset of lymphohematopoietic differentiation are not well characterized. Thus, the ES cell-derived Flk1 + population was further examined for the expression of cell surface molecules that might serve to identify the earliest lymphohematopoietic progenitors. CD105 (endoglin), an accessory receptor of the TGF-ß superfamily was expressed during early hematopoiesis from Flk1+ precursors (Flk1+ CD45- to Flk1- CD45+). Members of the TGF-ß superfamily have been implicated in mesoderm specification and hematopoiesis. Thus, a functional role for CD105 was assessed by examining the lymphohematopoietic differentiation of CD105-deficient ES cells in ES/OP9 cocultures. In the absence of CD105, myeloerythropoiesis was severely impaired in contrast to lymphopoiesis, which appeared to be only mildly affected. These findings should help to further our understanding of the mechanisms by which the bone marrow and thymic microenvironments induce or support lymphobematopoietic differentiation from mesodermal precursors.Ph.D